Isotype is a property of an antibody referring to those antigenic determinants that characterize the heavy and light chains of the immunoglobulin molecule into specific classes and subclasses. Isotype-specific antisera are prepared by immunizing an animal of one species with antibodies of a single immunoglobulin class from another species and then affinity purifying the resulting immune serum by adsorption against antibodies of the other classes to remove all components except those specific for the immunizing class.
The five immunoglobulin classes are distinguished structurally by differences in their heavy chain constant region. There are five major heavy chain classes, designated .alpha., .gamma., .delta., .epsilon. and .mu.. These heavy chain classes define the corresponding immunoglobulin classes IgA, IgG, IgD, IgE and IgM respectively. Some classes are divided into subclasses thereby indicating heavy chain constant regions which are distinct and closely related in their amino acid sequence. In the mouse the IgG class has subclasses .gamma.1, .gamma.2a, .gamma.2b and .gamma.3.
In addition to the class and subclasses defining the constant heavy chain region, there is also a classification based on the constant light chain region which is defined by comparisons in amino acid sequence. The classes defining the different light chains are classified as either .kappa. or .lambda. light chain. As with the heavy chain classifications, knowledge of the class aids in defining specific properties and physiological functions of the immunoglobulin molecule.
A knowledge of the isotype class and subclass of an antibody is necessary in order to more fully utilize the biochemical characteristics of a particular antibody. Characterization of a particular monoclonal antibody into its class and subclass serves as an additional means to identify the type of monoclonal antibody being produced by the hybridoma cell. In addition, knowledge of the immunoglobulin class aids in the development of purification schemes for the monoclonal antibody based on the particular physical and biochemical properties of its subclass. By utilizing specific properties of the antibody which are characteristic of the individual class, the ideal antibody can be chosen for use in specific immunologic assays.
When designing specific immunoassays or therapeutic applications of antibodies the different characteristics based on class can be exploited. There has been data reported which shows that IgG2a antibodies have more effect than the other subclasses in stimulating cellular immune response and as such may be useful in therapeutic studies. Certain IgM antibodies in addition to the IgG antibodies have been found to be good immunotoxins having an effect on immunotoxin action and biodistribution. In addition, certain subclasses have been found to be effective in initiating the complement reaction. On the other hand, some mouse IgG3 and IgM antibodies have a tendancy to precipitate out of solution and may therefore be unacceptable in many immunologic assays.
Determination of heavy and light chain isotypes is frequently used in the characterization of monoclonal antibodies; therefore, a variety of assays for the determination of light and heavy chain isotypes have been developed. The classical method for isotyping is known as Ouchterlony radial immunodiffusion wherein the solution containing the monoclonal antibody is placed in a round center well cut from an agarose base and isotype antisera are placed in surrounding wells in a hexagonal configuration. The antigen-antibody reaction is then noted by visible immunoprecipitin bands at the point of reactivity. This procedure, however, it slow and requires relatively large amounts of isotype-specific antisera. Another isotyping method is based on initial adsorption of monoclonal antibody to a solid phase antigen preparation. In this technique the immobilized antibody is then probed with radiolabeled isotype specific antisera or with unlabeled isotype-specific antisera followed by a radio-labeled or enzyme-tagged second antibody (Wagener, C. et al., 1983, J. Immunol. 130, 2308; Menard, S. et al., 1983, Cancer Research, 43, 1295).
An improvement on the use of the enzyme immunoassays (EIA) has recently been published (Bennett, F. et al., 1983, J. Immunol. Methods, 61, 201). In that article, the use of a dot immuno binding assay is described wherein up to ninety-six assays can be performed utilizing a single nitrocellulose sheet. A subsequent article (Horejsi, V. et al., 1983, J. Immunol. Methods, 62, 325.) describes the use of nitrocellulose membranes as carriers of antigen for rapid screening of specific monoclonal antibodies or as carriers of the monoclonal antibody for a determination of isotype. In all the referenced articles the antigen or monoclonal antibody is immobilized on a solid support whereon the EIA is performed. By binding the antigen or monoclonal antibody to the nitrocellulose paper first, numerous wash steps and additional reagents are necessary to achieve an accurate determination of the immunoglobulin class. Furthermore, it is often difficult to obtain suitable antigen in good quantity or to immobilize it successfully. The use of a specific antigen limits the generality of any isotype assay in which antigen immobilization is the initial step, since different antigens will be required if monoclonal antibodies of different specificity are to be tested.
Recently a method for detecting antibodies by means of reacting the antibody with antigen that has been spotted onto nitrocellose paper has been described (Herbrink, P. et al., 1982, J. Immunol. Methods, 48, 293). The antigen spot test is a highly sensitive assay for the detection of antibodies by utilizing a modification of the commonly used radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA). By spotting the antigen on the nitrocellulose paper the quantity of antigen required is reduced; however, a source of purified antigen directed to the monoclonal antibody of interest is still required in order to perform this assay. The method of Herbrink et al. does, however, provide a means of spotting onto nitrocellulose paper the various antigens thereby enabling the screening of numerous samples in a single assay. Bound antibodies are detected by incubating the paper containing the complex with either .sup.125 I-labeled protein A or using a second antibody conjugated to horseradish peroxidase. While the Herbrink, et al. paper does not teach the development of a method for isotype determination, it does serve as useful background information in the step-by-step development of successful immunologic assays based on the adsorption of an antigen-antibody complex onto a solid support.
Prior to the instant invention, the typing of monoclonal antibodies has required specific antigen and relatively large quantities of isotype specific antisera. The instant invention eliminates the need for specific antigen completely and is designed to be a rapid yet accurate method for determining isotype class without resorting to a multiplicity of steps or need for microtitration trays requiring numerous technical manipulations. By eliminating the need for specific antigen, a panel of monoclonal antibodies can be rapidly and accurately typed by using a solid support medium such as nitrocellulose strips with the adsorbed isotype antisera. When this probe is dipped in an antibody containing solution, e.g., spent hybridoma culture supernatant or mouse ascites fluid, then developed using a chromogenic substrate, the result is a visually detectable spot at the corresponding class and subclass for the specific antibody being tested.